Significance Airway gene transfer for the treatment of cystic fibrosis has progressed to early human clinical trials, however results of gene transfer following human aerosol administration have been very inconsistent using the vectors now available. These inconsistent results may in part be caused by the uneven distribution of aerosols in the lower airway. Objectives A method to evaluate the distribution in the airway of gene transfer vectors administered by aerosol inhalation would allow us to determine if the location of vector aerosol deposition is the primary factor responsible for the location of transgene expression, or if other host or vector specific effects determine the location of gene expression. The present pilot study was undertaken to compare specific airway imaging techniques in the evaluation of aerosol distribution using our quantitative aerosol administration system for aerosol administration of vectors and recombinant DNA for gene transfer. Results Aerosol exposure with 1 micron fluorescent microspheres results in deposition of readily identifiable microspheres in all lung lobes. Microspheres were visible in airways from the right anterior lung lobe after tissue was subject to critical point drying, cut, and imaged by fluorescent microscopy. Microspheres were more easily seen and the specific distribution by airways level identified with fluorescent dissecting microscope when the perfusion fixed lung lobe was microdisected to reveal the airway prior to imaging. The latter technique appears to be superior for determining the specific distribution of material in the bronchiolar airways. Future Directions The technique of airway microdissection and fluorescent imaging will be used to compare transgene expression and aerosol distribution in the same lung lobe. KEYWORDS aerosol gene transfer, cystic fibrosis